The NCMI is dedicated to the advancement of cryo-electron microscopy and tomography methodology for structure determination of macromolecules, molecular machines and cells in their various functional states at the highest possible resolutions. Having completed 10 C-alpha backbone traceable cryo-EM structures of molecular machines during the current grant period, we are well poised to tackle the next set of challenging structural biology problems. Our technology research development will focus around optimization of cutting edge instrumentation, data collection strategy, data management, image processing, modeling and visualization from electron images recorded from two unique combinations of instrumentation: (i) a 300 kV electron microscope with a direct electron detector and an in-column energy filter and (ii) a 200 kV electron microscope with a direct electron detector, an in-column energy filter and a Zernike phase contrast optics. Our technology development is led by 10 driving biological projects and also synergizes with 10 user projects that together include animal, plant and bacterial viruses, apoptosis-causing protein machine, chaperonin-substrate complex, membrane ion channels, nuclear receptor-coactivator complex, small RNA, oncogene protein complex, lipoproteins, amyloid protein aggregates, neuronal cells, virus-infected cells and mammalian cells related to cancer and eye diseases. Specifically, our Center will focus on 3 technology research and development projects: (i) Characterize and determine the optimal utilization of direct electron detectors and Zernike phase optics; (ii) Extend the structural determinations of biochemically purified molecular machines beyond current resolution limits; (iii) Develop novel methodologies for analyzing subcellular structures i cells by cryo-ET. We will proactively identify new projects from NIH-funded investigators across the US. We will continue our rigor in disseminating our software, experimental and computational protocols via workshops and web seminars. We will maintain an engaging advisory board to critique our progress and guide our strategic planning annually. Our efforts will extend cryo-EM/cryo-ET capabilities to fill the information gaps between x-ray crystallography, NMR and optical microscopy from nanometer to atomic resolutions.